Microtubules play an essential role in the subcellular organization, polarization, and motility of mammalian cells. During muscle differentiation they are dramatically reorganized, and are assumed to be responsible for the redistribution of essential organelles such as the Golgi complex. We have previously shown that in adult muscle in vivo their organization is fiber-type dependent and is plastic in response to muscle activity. Microtubules may play a role in the initial organization of contractile proteins and in vivo may be involved in muscle atrophy. Yet we understand practically nothing of their regulation in muscle.[unreadable] [unreadable] We are currently investigating the function and regulation of microtubules during muscle differentiation by challenging the muscle cell line C2 to differentiate in the presence of pharmacological agents or of cDNA constructs that affect microtubule stability or integrity. This work has led us to identify the kinase GSK3-beta, which regulates microtubule stability, as playing a role in the reorganization of the microtubule-organizing center during muscle differentiation. We have also identified a new role for the microtubule-associated protein EB1 which, in non-muscle cells, is responsible for microtubule stabilization. Having observed that the expression of a dominant-negative partial construct of EB1 prevents the normal elongation of muscle cells, we used siRNAs and shRNAs to create muscle cell lines depleted of EB1. Unexpectedly, these cells fail not only to elongate but also to fuse and some of them fail to express the myogenic proteins characteristic of muscle differentiation. We have observed that the effect of EB1-targeted shRNAs on myogenesis are linked to their ability to suppress not only the 30 kDa form of EB1 but also a 20 kDa, muscle-specific isoform that has not been reported before. Our goals are to identify this EB1 isoform and to follow these leads and continue to identify signaling pathways regulating the reorganization of muscle cells during differentiation.[unreadable] [unreadable] Intermediate filaments interact with microtubules and the two systems are partially colocalized in muscle in vivo. In order to identify whether intermediate filaments are necessary for the normal organization of the microtubule network, we are using a line of transgenic mice in which desmin, the main intermediate filament protein of muscle, is absent. We have indeed found that microtubules and subcellular organelles are affected in muscles of the desmin-null mice. In particular, we have observed that the nuclei lose their normal regular disposition along the muscle fibers and form aggregates alongside the blood vessels that irrigate the muscle fibers.